Polyhydroxyalkanoate production methods and systems for same

ABSTRACT

Several embodiments of the invention relate generally to a system and methods for the treatment of gaseous emissions comprising methane and one or more non-methane compounds that can influence the metabolism of methane-oxidizing microorganisms. In several embodiments, there is provided a system and methods for the treatment of methane emissions through the use of methanotrophic microorganisms to generate functionally consistent and harvestable products. Certain embodiments of the invention are particularly advantageous because they reduce environmentally-destructive methane emissions and produce harvestable end-products.

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/286, 274, filed May 23, 2014, which is a continuation ofU.S. patent application Ser. No. 13/310,542, filed Dec. 2, 2011, nowissued as U.S. Pat. No. 8,735,113, the entire disclosures of each ofwhich is incorporated by reference herein.

BACKGROUND

1. Field of Invention

Several embodiments of the invention relate generally to a system andmethods for the treatment of methane emissions, and in severalembodiments, to systems and/or methods for the treatment of methaneemissions through the use of methanotrophic microorganisms.

2. Description of the Related Art

Methane emissions, or methane off-gases, are generated by a variety ofnatural and human-influenced processes, including anaerobicdecomposition in solid waste landfills, enteric fermentation in ruminantanimals, organic solids decomposition in digesters and wastewatertreatment operations, and methane leakage in fossil fuel recovery,transport, and processing systems. As a particularly potent greenhousegas, methane emissions are estimated to be responsible for about twentypercent of anthropogenic global warming, and thus represent asignificant environmental problem. Accordingly, there have been numerousefforts in the past to remediate, control, and/or otherwise treatmethane emissions from a variety of sources.

SUMMARY

Several embodiments disclosed herein are directed to processing methanethat is emitted from landfills, coal mines, wastewater treatment plants,manure digesters, agricultural digesters, compost heaps, enclosedagricultural feedlots, and leaking or otherwise emitting petroleumsystems.

Atmospheric methane emissions are estimated to account for about twentypercent of global warming. In addition to the environmentallydestructive effect of methane emissions, such emissions represent wastedcarbon and energy, as methane could theoretically be used to generateenergy (e.g., heat homes or drive combustion-based processes), providefuel, and, or serve as a carbon input for the generation of usefulproducts, such as chemicals, plastics, fuels, and other products.

Several methods are known for the treatment of natural andhuman-influenced methane emissions. Used in conjunction with methaneemissions collection methods, such as landfill gas extractionwells/blowers and coal mine methane ventilation systems, the treatmentof air containing captured methane emissions includes the use ofturbines, microturbines, engines, reverse-flow reactors, fuel cells, andboilers to convert methane emissions into heat and/or electricity. Othermethods for the treatment of methane emissions include the conversion ofmethane emissions into pipeline-quality, liquefied, or compressednatural gas.

The utilization of methane emissions for the production of fuel, heat,and/or electricity is described by a number of patents, including U.S.Pat. Nos. 5,642,630, 5,727,903, 5,842,357, 6,205,704, 6,446,385, and6,666,027, each of which is herein incorporated by reference in itsentirety. U.S. Pat. No. 5,642,630 describes the use of landfill gas toproduce high quality liquefied natural gas, liquefied carbon dioxide,and compressed natural gas products. U.S. Pat. No. 5,727,903 describesthe use of landfill gas to create vehicle grade fuel. U.S. Pat. No.5,842,357 describes the use of landfill gas to create high grade fueland food-grade carbon dioxide. U.S. Pat. Nos. 6,205,704 and 6,446,385describe the use of landfill gas to provide heat, electricity, and/orcarbon dioxide to enhance greenhouse operations. U.S. Pat. No. 6,666,027describes the use of off-gas from landfills and digesters to powerturbines for electricity generation.

Although each of these methods can treat methane emissions under certainspecific conditions, none are known to be economically and/ortechnologically feasible in most practical contexts, including under arange of sub-optimal methane-in-air conditions. For example, theeffectiveness of such methods is reduced or eliminated entirely underconditions where the flow rate, concentration, or purity of methane gasemissions is variable (e.g., unpredictable, low, and/or otherwiseunfavorable), as is commonly the case when using natural sources ofmethane emissions or when considering methane emissions from sourcessuch as landfills, dairy operations, wastewater treatment plants, orindustrial off-gases.

Methane-utilizing, or methanotrophic, microorganisms are known in themicrobiology art for their capacity to grow and reproduce using methaneas a source of carbon and/or energy. These microorganisms are known togrow in a wide range of diverse methane availability conditions.Accordingly, methanotrophic microorganisms have been proposed in thepast as a potential tool for the remediation of methane emissions,particularly in conditions where other treatment methods aretechnologically and/or economically unfeasible.

Two methods have been proposed for the utilization of methanotrophicmicroorganisms to treat methane emissions. In one proposed process,methanotrophic microorganisms are naturally present or purposefullysituated in high-methane emissions environments, such as landfill coversor coal mines. The methanotrophic microorganisms are provided withgrowth-stimulating nutrients, such as oxygen, water, or mineral salts,to encourage increased microbial methane emission uptake rates. Thismethod may be carried out using nutrient injection methods such as airor water sparging to induce increased methanotrophic growth andoxidation rates in high emissions environments. U.S. Pat. No. 6,749,368,for example, describes methanotrophic microorganisms that are placed inan aerated soil cover above a municipal landfill in order to oxidize andreduce methane emissions.

In a second proposed process, air containing methane emissions isdiverted into an environment containing methanotrophic microorganisms inorder to cause the microbial breakdown of methane emissions. This methodmay be carried out by diverting air containing methane emissions into abiofiltration column containing methanotrophic microorganisms, water,and a microorganism growth medium, whereby electricity, water, nitrogen,trace minerals, and other materials are continuously added to andconsumed by the system in order to effect the microbial breakdown ofmethane emissions.

Both of these methanotrophic treatment techniques cannot effectively orefficiently reduce methane emissions. Indeed, the application of theseprocesses has been almost entirely precluded in practice because bothhave continuous requirements for costly materials, such as electricityand minerals, yet neither generates economic benefits to recover thosecapital costs of methane emission treatment. Thus, the use ofmethanotrophic microorganisms for the treatment of methane emissionswhich generate no commercially useful products is simply too costly tooperate and sustain over time. Prior to the Applicants' discovery, nomethods were known to enable the treatment of methane emissions whereincommercially useful (e.g., high value and having consistent functionalproperties) products could be generated, and, accordingly, theutilization of methanotrophic microorganisms for the treatment ofmethane emissions has been precluded in practice.

Accordingly, there exists a significant need to develop methods andsystems that enable methanotrophic methane emissions treatment to becarried out in a manner that generates commercially useful (e.g., highvalue and having consistent functional properties) products, therebyrendering the process commercially viable and technologically,financially, and logistically sustainable.

Several embodiments of the present invention address the need for asystem that enables biological methane emissions treatment to generateharvestable, e.g., commercially useful, products, and thus betechnologically, financially, and logistically sustainable and viable.Prior to embodiments of Applicants' invention as disclosed herein,gaseous emissions comprising methane have never been used in conjunctionwith methanotrophic microorganisms to reduce the environmentallydestructive impact of methane emissions while simultaneously creating aharvestable product (or products) from that methane.

In several embodiments, the gaseous emissions (which comprise someamount of methane) from landfills, coal mines, agricultural sites, orpetroleum sites are captured and conveyed to a bioreactor containingmethanotrophic microorganisms. In some embodiments, the gaseousemissions do not need to undergo substantial purification. In stilladditional embodiments, no purification of the gaseous emissions isrequired, though in other embodiments, purification is optionallyperformed. The microorganisms use the methane as a source of carbon orenergy and, in some embodiments, produce useful end-products, such aspolymers or plastics, with physical, chemical, and performance qualitiesthat can used for commercial purposes, e.g., to replace oil-basedplastics. The polymers or plastics can then be used to synthesizevarious types of materials, including, but not limited to, biodegradableplastic parts. In some embodiments, the polymers can be used to replacea wide range of oil-based plastics, such as polypropylene, polyethylene,and polystyrene, due to the physical properties of the plastics, whichare also biodegradable. Thus, some preferred embodiments of theinvention offer a tremendous benefit to the environment in at least twoways: first, methane emissions are substantially reduced on the frontend, thereby sequestering carbon that would have otherwise been emittedinto the air, and second, a biodegradable polymer that can be used toreplace oil-based plastic is produced in useful quantities as theend-product, thereby reducing the use of non-renewable oil.

The term “gaseous emission” as used herein shall be given its ordinarymeaning and shall also refer to off-gases and/or gases produced,generated, or emitted by natural and/or human-influenced processes,including anaerobic decomposition in solid waste landfills, organicdecomposition in digesters and wastewater treatment operations,agricultural sites, and in fossil fuel recovery, transport,distribution, delivery, and processing systems.

Although the prior art recognized that methanotrophic organisms coulduse methane to produce polymers, the prior art did not disclose, teach,or suggest an effective method by which destructive gaseous emissionsthat comprise methane could be used to produce polymers (or otherharvestable and useful products as disclosed herein) from gas streamscomprising methane and non-methane compounds or substances that impactthe metabolism of methanotrophic microorganisms, as the case may be thecase with industrial or municipal methane emissions. Prior toApplicants' invention, the production of commercially useful polymers(or other products) by methanotrophic organisms from non-pure methaneemissions from sources such as, e.g., landfill gas, was not feasible,because the process would generate polymers or proteins (or otherbiological products such as those disclosed herein) having a wide,inconsistent, and unpredictable range of functional properties based onthe varied type and varied concentration of non-methane impuritiescontained in the methane emissions streams (as well as the variation inthe concentration of methane itself). In contrast, several embodimentsof the present invention do not require artificial laboratory grademethane (which is essentially pure methane) to produceharvestable/commercially useful polymers. Instead, environmentallydestructive gases that are already present in the environment,particularly those gases comprising variable concentrations of methaneand non-methane compounds or substances that impact methanotrophicmetabolism, are used as the source of methane.

Therefore, in several embodiments there are provided methods forproducing a polyhydroxyalkanoate (PHA) in a culture of methanotrophicmicroorganisms, comprising providing a gas comprising methane and one ormore non-methane substances, providing a culture of methanotrophicmicroorganisms and a microorganism culture medium comprising at least afirst essential nutrient and a second essential nutrient, exposing theculture to the gas, and controlling the concentration of the firstessential nutrient in the culture medium to a concentration sufficientto induce the methanotrophic microorganisms to produce particulatemethane monooxygenase (pMMO) and/or soluble methane monooxygenase(sMMO), and controlling the concentration of the second essentialnutrient, wherein the control of the second essential nutrient causesthe methanotrophic microorganisms to produce the PHA.

In addition to the methods disclosed herein, there is also provided asystem for producing PHAs with consistent functional properties from aculture of methanotrophic microorganisms. In several embodiments, thesystem comprises a culture of methanotrophic microorganisms capable ofmetabolizing gas comprising methane and one or more non-methanesubstances, a microorganism culture medium comprising at least a firstessential nutrient and at least a second essential nutrient, abioreactor for culturing the methanotrophic microorganisms in thepresence of the microorganism culture medium, and a conveyer thatconveys the gas from the source of gas into the bioreactor, therebyexposing the methanotrophic microorganisms and the microorganism culturemedium to the gas. In several embodiments, the concentration of thefirst essential nutrient is controlled to a concentration sufficient toinduce the methanotrophic microorganisms to produce particulate methanemonooxygenase (pMMO) and/or soluble methane monooxygenase (sMMO) and theconcentration of the essential nutrient is controlled to a concentrationsufficient to cause the methanotrophic microorganisms to generate PHA.In several embodiments, the control of the concentration of the firstessential nutrient and the second essential nutrient make up aproduction cycle comprising methane monooxygenase production followed byPHA production. In several embodiments, the system is configured torepeat the production cycle to induce at least a first production cycleand a second production cycle, wherein the functional properties of PHAgenerated in the first production cycle are substantially similar to thefunctional properties of PHA generated in the second production cycle.

In several embodiments, the PHA is selected from the group consisting ofpolyhydroxybutyrate, polyhydroxybutyrate-covalerate (PHBV),poly-4-hydroxybutyrate (P4HB), polyhydroxyhexanoate (PHHx), andpolyhydroxyoctanoate (PHO).

In several embodiments, certain individual steps or combinations ofsteps can be categorized based on the result achieved after performanceof those steps. For example, in certain embodiments, the methodcomprises a microorganism growth phase, a monooxygenase productionphase, and PHA production phase. Additional embodiments comprise, forexample, a harvesting phase. In some embodiments, or more steps can beperformed a single time or, in other embodiments, can be repeatedmultiple times. For example, in some embodiments induction of pMMO orsMMO production is followed by induction of PHA production, whichcomprises a production cycle. In several embodiments, multipleproduction cycles are performed (e.g., induction of pMMO or sMMOproduction followed by induction of PHA production multiple times),thereby resulting in at least a first production cycle and a secondproduction cycle. The repeated performance of such production cyclesallows the enhanced production of PHA (e.g., greater concentrationsand/or more similar functional properties). In some embodiments therepetition of such production cycles increases the overall efficiency ofthe process (e.g., a greater percentage of the methane from the gas isconverted to useful and harvestable and product such as PHA).

Consistency in the characteristics of a manufactured product aregenerally desirable, in that a user of such a product can expect thatthe behavior of the product will be similar from use to use (e.g., frombatch to batch). This consistency reduces inefficiencies in using theproduct because a more standardized protocol for use of the product canbe developed and implemented. Moreover, consistency increases the user'sability to obtain reproducible results over time (e.g., a productsubsequently produced by the user is likely to also be more consistentover time). The methods and systems disclosed herein are advantageous inthat they allow the production of products that are have consistentfunctional properties, even in spite of microbial growth conditions thatmay vary substantially over time. In several embodiments, polymers areproduced using the methods and/or systems disclosed herein. In someembodiments, the functional characteristics of the generated polymersinclude, but are not limited to molecular weight, polydispersity and/orpolydispersity index, melt flow and/or melt index, monomer composition,co-polymer structure, melt index, non-PHA material concentration,purity, impact strength, density, specific viscosity, viscosityresistance, acid resistance, mechanical shear strength, flexularmodulus, elongation at break, freeze-thaw stability, processingconditions tolerance, shelf-life/stability, hygroscopicity, and color.In several embodiments, consistency in more than one of these functionalproperties is achieved. For example, in some embodiments, consistentmolecular weight, polydispersity, and combinations thereof are achieved.

The generation of a product, such as PHA, that has consistent functionalcharacteristics enables the end user of the PHA to have greaterassurance that the PHA will perform in a similar fashion each time it isused, which reduces inefficiencies in manufacturing or other processthat utilize that PHA and also may increase the quality, performance,and/or value of a subsequently produced item.

In several embodiments, the molecular weight of the PHA produced in afirst production cycle differs from the molecular weight of the PHAproduced in a subsequent production cycle by less than 50%. Inadditional embodiments, the molecular weight of PHA produced indifferent production cycles differs by 25% or less, 20% or less, 10% orless, 5% or less, or 1% or less. In several embodiments, the molecularweight of PHA produced ranges from about 100 to about 5,000,000 Daltons.In several embodiments, the molecular weight of PHA produced ranges fromabout 100,000 to about 2,500,000 Daltons. Other molecular weight rangesare achieved in certain other embodiments.

In several embodiments, the polydispersity of the PHA produced in afirst production cycle differs from the polydispersity of the PHAproduced in a subsequent production cycle by less than about 75%. Insome embodiments, the polydispersity of the PHA produced in variousproduction cycles differs by less than about 50%. In some embodiments,the polydispersity between production cycles differs by about 25% orless, about 20% or less, about 10% or less, about 5% or less, or about1% or less. In some embodiments the polydispersity of PHA produced in afirst production cycle is indistinguishable from that of PHA produced indifferent production cycle. In several embodiments, the polydispersityranges from about 0.1 to about 5.0, including about 0.1 to about 0.5,about 0.5 to about 1.0, about 1.0 to about 1.5, about 1.5 to about 2.0,about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5,about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0,and overlapping ranges thereof.

In several embodiments, the concentration of the pMMO and the sMMOproduced in the microorganisms in a first production cycle differs byless than about 75% from the total concentration of the pMMO and thesMMO produced in the microorganisms in a subsequent production cycle. Inadditional embodiments, concentration of the pMMO and the sMMO producedin a first production cycle differs from the concentration produced in adifferent production cycle by less than about 50%, by less than about25%, by less than about 10%, or by less than about 1%.

In several embodiments, the one or more non-methane substances have theability to impact the metabolism of the methanotrophic microorganisms.Advantageously, however, the method allows the production of PHA havingconsistent functional properties despite the potential impact that suchsubstances have on the metabolism of the microorganisms. In severalembodiments, the one or more non-methane substances are selected fromthe group consisting of methanol, acetone, acetate, formate,formaldehyde, hydroxyalkanoates, hydroxybutyrate, octanoic acid,octanol, carbon dioxide, nitrogen, oxygen, di-oxygen, di-nitrogen,water, water vapor, argon, ethane, propane, butyrate, butyric acid,hexanoic acid, hexanol, heptanoic acid, heptanol, pentane, pentanoicacid, and volatile organic compounds.

In several embodiments, the culture comprises two or more species ofmethanotrophic microorganisms. In several embodiments, the two or morespecies are selected based on complementary characteristics. Forexample, a first species may be selected based on its ability tometabolize low concentrations of methane while a second species may beselected based on its ability to maintain metabolic functionality in theface of impurities in the culture environment. In some embodiments, byplacing culturing two such species together a more efficient processingof a gas comprising methane and one or more non-methane compounds (suchas, for example landfill gas or gas from decomposition in digesters andwastewater treatment operations) is achieved.

At least in part, the control of the concentration of essentialnutrients in the culture media allows the metabolic synchronization ofthe microorganisms within the culture such that functionally consistentproducts are generated. In several embodiments, the first and the secondessential nutrients comprise one or more of carbon, hydrogen, nitrogen,oxygen, phosphorus, potassium, calcium, sodium, chlorine, methane,carbon dioxide, magnesium, iron, copper, sulfate, manganese, boron,zinc, aluminum, nickel, chromium, cobalt, or molybdenum.

In some embodiments, combinations of essential nutrients are controlled.In some embodiments, controlling of the first essential nutrient or ofthe second essential nutrient comprises increasing the concentration offirst essential nutrient or of the second essential nutrient. In someembodiments, the controlling of the first essential nutrient or of thesecond essential nutrient comprises decreasing the concentration offirst essential nutrient or of the second essential nutrient. In someembodiments, the first and second essential nutrients are the samenutrient.

In still additional embodiments, a first essential nutrient may beincreased while a second essential nutrient may be decreased. In certainembodiments, controlling the concentration comprises maintaining theconcentration within a certain range (as opposed to increasing ordecreasing the concentration of that nutrient). In such embodiments,maintenance may be achieved by adding sufficient amounts of thatnutrient to offset consumption (e.g., metabolism) of the nutrient by themethanotrophic microorganisms. Likewise, the overall volume of theculture media can be adjusted, for example by adding or removing water,such that the concentration of a particular nutrient is maintained. Insome embodiments, maintenance of the concentration of essential nutrientcomprises limiting variation in the concentration to less than about50%. In some embodiments, the variation is limited to less than about25%, less than about 20%, less than about 15%, less than about 10%, orless than about 5%. Depending on the nutrient, in some embodimentsgreater or lesser degrees of variation are allowable.

Moreover, several embodiments of the present invention are particularlyadvantageous because gaseous emissions comprising low concentrations ofmethane can be used, rather than pure (high methane concentration, forexample, concentrated methane and/or methane with low or non-existentcontaminants) methane. Although certain turbine systems are capable ofconverting gaseous emissions into energy, the concentration of methanemust be high. Likewise, although certain fuel cells can use methane inlow concentrations, gaseous emissions such as those employed in themethods disclosed herein cannot be used because the fuel cells requiremethane that must be substantially pure.

Advantageously, in several embodiments, gaseous emissions comprisingmethane in a concentration of less than about 95%, less than about 80%,less than about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, and less than about 1% can be used. Thus,several embodiments of the present invention are particularly useful forolder landfills, which may produce methane emissions with methaneconcentrations of about 0.1% to less than about 20% of total gaseousemissions as they age. Likewise, several embodiments of the presentinvention are particularly useful for coal mines, which may producemethane in concentrations of less than about 5% of total gaseousemissions, and in some cases about 1% methane. Thus, without the benefitof certain preferred embodiments of Applicants' invention, these sourcesof methane (and other gases) pollution—which alone produce methane as asmall part of their total gaseous emissions—cumulatively contributesignificantly to the total amount of methane in the environment and thusultimately to the greenhouse effect.

As discussed previously, methane emissions are anenvironmentally-destructive material and represent a largely unusablesource of energy and carbon. In several embodiments of the invention,methane emissions are used to produce a useful end-product (or products)that can be used or sold for use, thereby providing an economicincentive to a methane emissions reduction effort. While in severalembodiments, the harvestable and useful end-product is a polymer (e.g.,PHA), additional harvestable goods include, but are not limited to, themicroorganism culture itself, products or compositions that can beobtained from the microorganism culture (e.g., a harvested enzyme, suchas methane monooxygenase, including particulate methane monooxygenaseand/or soluble methane monooxygenase), the oxidative products of methanemonooxygenase, methanol, carbon dioxide, or combinations thereof. Thus,in some embodiments, gaseous emissions comprising methane are used togrow a microorganism culture to a density and quality that is capable ofbeing harvested and commercially used, sold, or traded. In oneembodiment, the microorganism culture and/or the products createdthereby can be used, for example, as a nutrition source for livestock,exhibiting a consistent polymer functional profile, or a biodegradableplastic exhibiting commercially useful properties, such as consistentmolecular weight and consistent molecular composition. In someembodiments, the end-product is a culture of microorganisms, or theproducts generated by those microorganisms (e.g., PHA thermoplasticpolymers), or combinations thereof.

In several embodiments of the invention, methane emissions are processedto produce useful and harvestable products. These products include, butare not limited to: protein-rich or polymer-rich biomass,polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB),polyhydroxybutyrate-valerate (PHB/V), particulate or soluble methanemonooxygenase (pMMO or sMMO, respectively), vaccine derivatives,enzymes, polymers, cellular materials, formaldehyde, and methanol,propylene oxide, or combinations thereof. In several embodiments of theinvention, methanotrophic microorganisms can be manipulated andprocessed to generate useful (e.g., defined, consistent, andharvestable) goods in sterile, semi-sterile, and/or non-sterileconditions.

In several embodiments of the invention, a culture of suitablemethanotrophic microorganisms is provided for the efficient, effective,and commercially viable treatment of methane emissions. The prior artgenerally criticizes the use of methanotrophic organisms to treatmethane emissions because, as described above, such a process wasthought to generate an varied and unpredictable, inefficient, andunreliable culture of microorganisms and/or products. For example, theprior art teaches that bioremediation and biofiltration generates amicroorganism conglomerate that is non-specific, non-defined, and/orhighly variable over time according to shifts in nutrient availability,air contamination, species interaction, and so on. As emphasized in U.S.Pat. No. 6,599,423, “prior art teaches that ex situ biofilters andbioreactors are akin to microorganism zoos, with the microorganismcultures naturally adapting, dominating, and maintaining themselvesaccording the various compounds, food sources, and contaminants presentor fed to the biodegradation media . . . changes, adaptations, anddominance of certain cultures will occur even in such isolated andinoculated cultures after operation begins and the biofilters orbioreactors are subjected to complex mixtures of food sources,contaminants, and microorganisms present in the natural environment.”

As discussed above, such changes that occur in the culture over time canlead to corresponding changes in the products that are harvested fromthe culture. Absent methods of control, as disclosed herein, microbialcultures and the byproducts generated from the growth thereof arecreated in a variable, non-specific, unpredictable, speculative, orotherwise non-useful manner. Product variability is generally undesired,and by contrast, in several embodiments of the present invention,systems for using and methods of using microorganisms in a highlycontrolled manner for the treatment of gaseous emissions are provided.In one embodiment, the invention provides a mechanism to cause a cultureof methanotrophic microorganisms to produce a stable or controlledmetabolic end-product (e.g., PHA) despite variability in concentrationsof different methanotrophic microorganism species, or metabolicadaptations amongst the methanotrophic microorganisms in the culture asculture medium conditions change. Specifically, in one embodiment, theinvention enables the controlled and stable production of PHA polymersby methanotrophic microorganisms in a culture medium despite changes inthe culture and/or culture medium caused by the presence of non-methanecompounds or substances that influence the metabolism by methanotrophicmicroorganisms. Specifically, by controlling the production of sMMO orpMMO in the methanotrophic microorganism culture, the metabolic statusof the culture and/or the sources of carbon that may be metabolized bythe culture are controlled, which enables the simultaneous (orsubsequent) induction of PHA polymer synthesis by the methanotrophicmicroorganisms.

In several embodiments of the invention, an apparatus or system forprocessing methane emissions and producing harvestable products isprovided. In one embodiment, the system comprises (i) a source ofgaseous emissions, wherein the gaseous emissions comprise methane and atleast one non-methane compound, (ii) methanotrophic microorganisms thatuse methane as a source of carbon or energy, (iii) a bioreactor thatencloses or contains the methanotrophic microorganisms, and (iv) aconveyer that conveys the gaseous emissions into the bioreactor, therebyexposing the methanotrophic microorganisms to the gaseous emissions andcausing the methanotrophic microorganisms to produce a harvestableproduct after using the methane as a source of carbon or energy, whereinthe conditions within the bioreactor are controlled to enable theproduction of one or more harvestable products.

In accordance with several embodiments of the invention, a novel methodfor enabling the viable treatment of air containing methane emissions isprovided. In one embodiment, methanotrophic microorganisms and aircontaining methane emissions are mutually-exposed to cause or enableharvestable (e.g., commercially useful) product formation. Theharvestable product may be used or sold. In additional embodiments ofthe invention, air containing methane emissions may be used to createsingle cell protein, enzymes, polymers, or other bio-based products in amanner that enables harvest of commercially useful product. In stillfurther embodiments, more than one of the harvestable products disclosedherein are produced together (e.g., sequentially or simultaneously).

In some embodiments, the invention comprises a method of processingmethane emissions for the production of a harvestable product,comprising: providing a gaseous emission comprising methane andproviding methanotrophic microorganisms, exposing the methanotrophicmicroorganisms to the gaseous emission, wherein the methanotrophicmicroorganisms use at least a portion of the methane as a source ofcarbon or energy, and controlling the concentration of copper and one ormore essential nutrient, such as nitrogen, oxygen, magnesium,phosphorus, calcium, sodium, sulfate, methane, carbon dioxide, iron,manganese, zinc, cobalt, chromium, aluminum, boron, and/or molybdenum,wherein the methanotrophic microorganisms produce a harvestable (e.g.,commercially useful, functional, defined, and/or consistent) productafter using the methane as a source of carbon or energy.

In several embodiments, the harvestable product comprises a polymer(such as polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), andpolyhydroxybutyrate-valerate (PHB/V)). In another embodiment, theharvestable product comprises one or more of the following:microorganism biomass, methane monooxygenase (particulate or soluble),protein-rich biomass, enzymes, and cellular contents. In additionalembodiments, the harvestable product comprises a quantifiable reductionin methane emissions. In still further embodiments, the methods andsystems disclosed herein result in combinations of one or more of theharvestable products disclosed herein.

In several embodiments, the gaseous emission comprises a gas selectedfrom the group consisting of one or more of the following: carbondioxide, ammonia, nitrous oxide, air, nitrogen, and ozone. In oneembodiment, the gaseous emission comprises unpurified landfill gas,partially purified landfill gas, anaerobically-generated gas, naturalgas, or gas from the production, generation, distribution, and/orprocessing of fossil fuels. In one embodiment, one or more impuritiesare removed from the gaseous emission. In another embodiment, thegaseous emission is disinfected using ultraviolet light. In severalembodiments, multiple sources of gaseous emissions are used (e.g., thegaseous emissions are combined).

In one embodiment, the invention comprises harvesting the harvestableproduct for commercial or industrial sale or use.

In one embodiment, the invention comprises substantially reducing oreliminating (or otherwise adjusting, controlling, or depleting) theconcentration of nitrogen available to the methanotrophicmicroorganisms. In some embodiments, other elements, nutrients, orcompounds that are available to the methanotrophic microorganisms arereduced, substantially reduced, depleted, eliminated, or otherwiseadjusted or controlled (in addition to or in place of the nitrogen) inorder to induce product formation. Such elements and compounds include,but are not limited to carbon, oxygen, magnesium, phosphorus, phosphate,potassium, sulfate, sulfur, calcium, boron, aluminum, chromium, cobalt,iron, copper, nickel, manganese, molybdenum, sodium, nitrogen, nitrate,ammonia, ammonium, urea, amino acids, methane, carbon dioxide, and/orhydrogen.

In several embodiments, the invention comprises using gaseous emissionshaving methane concentrations in the range of about 0.1% to about 10%,in the range of about 10% to about 20%, and at concentrations greaterthan about 20%. In another embodiment, the methane concentration is lessthan about 5%. In yet another embodiment, the methane concentration isbetween about 30% to about 60% of the total gaseous emissions, andcarbon dioxide concentration is about 30% to about 60%. The latternumbers are typical of certain landfill emissions.

In several embodiments, the gaseous emission is generated by one or moreof the following: coal mine, wastewater treatment operation,agricultural digester, enclosed feedlot, petroleum transport system, andpetroleum recovery system.

In several embodiments, the microorganisms comprise naturally-occurringor genetically-modified microorganisms, or a combination thereof, thatuse methane as a source of carbon or energy for growth or reproduction.The methanotrophic microorganisms may include one or more of thefollowing: Methylococcus capsulatus, Alcaligenes acidovorans, Bacillusfirmus, and Bacillus brevis.

In additional embodiments, the gaseous emission comprises, in additionto methane, a non-methane compound, wherein the non-methane compound isan organic or inorganic compound or material or substance. In anotherembodiment, the gaseous emission comprises a non-methane compound orsubstance such as toluene, benzene, methanol, propylene, alkenes,alcohol, ether, ethane, propane, butane, isobutane, formaldehyde, andtrichloroethylene, or a combination thereof. Non-methane compounds orsubstances may also include non-methane gases such as carbon dioxide,oxygen, nitrogen, ammonia, nitrous oxide, and ozone.

In several embodiments, the non-methane compound or substance ismetabolized, consumed, or used by the methanotrophic microorganisms. Inseveral embodiments, the non-methane compound or substance is producedintracellularly. In several embodiments, the non-methane compound orsubstance is present in the growth medium at a concentration of lessthan 100,000 part per million (ppm), less than 10,000 ppm, less than1000 ppm, less than 100 ppm, less than 10 ppm, less than 1 ppm, lessthan 100 part per billion (ppb), less than 10 ppb, or less than 1 ppb.

In several embodiments, the invention comprises reducing theconcentration of methane to a concentration compliant with applicableenvironmental regulations or laws. In the United States, for example,preferred embodiments of the invention reduce methane to concentrationssuggested or mandated by local, state, and federal EPA guidelines.

In several embodiments, the present invention comprises a method ofproducing a commercially useful biodegradable polymer from landfill gas.In some embodiments, the method comprises obtaining landfill gas,wherein the landfill gas comprises methane, enclosing the landfill gasin a bioreactor containing methanotrophic microorganisms and growthmedium, and inducing the methanotrophic microorganisms to producebiodegradable polymer by controlling, substantially reducing, ordepleting the growth medium of one or more compounds, elements, ornutrients necessary for growth of the methanotrophic microorganisms. Asdisclosed herein, all of the various components (including elements,compounds, liquids, gases, solids, and other compositions) of a culturemedium can be considered an essential nutrient, given that they supportthe growth of the microorganisms (such as carbon, oxygen, magnesium,phosphorus, phosphate, potassium, sulfate, sulfur, calcium, boron,aluminum, chromium, cobalt, iron, copper, nickel, manganese, molybdenum,sodium, nitrogen, nitrate, ammonia, ammonium, urea, amino acids,methane, carbon dioxide, and/or hydrogen). In one embodiment, the methodfurther comprises harvesting the biodegradable polymer (or otherproduct).

In several embodiments of the present invention, a system to reducemethane emissions or gaseous emissions comprising methane is provided.In some embodiments, the emissions are produced by landfills, wasteprocessing sites, coal mines, and/or other similar systems created byhumans.

Thus, in accordance with several embodiments, methane-containing gaseousemissions are used as a source of carbon and/or energy for the inductionof a methane-driven process and/or for the production of methane-derivedmaterials, such as methane-utilizing microorganisms, heat, and/orelectricity.

As discussed previously, methane is an environmentally-destructivematerial and previously unusable source of energy, which, according toseveral preferred embodiments of the invention, is used to produce auseful end-product that can be used or sold for use, providing aneconomic incentive for methane emissions reduction efforts on variousscales and from various sources. In one embodiment, the end-product isheat. In another embodiment, the end-product is fuel. In yet anotherembodiment, the end-product is electricity. In yet another embodiment,the end-product is another form of energy. In further embodiments, theend-product is the culture of microorganisms or a byproduct isolatedfrom the culture.

The term “air” as used herein shall be given its ordinary meaning, andshall include all airborne and gaseous components of air that have beencontacted with or comprise methane, as well as ammonia gas, dust,microorganisms, and/or other airborne materials that may be present inthe air.

In several embodiments, the term methane-consumption means shall begiven its ordinary meaning and shall also include any means by which themethane is oxidized, consumed, and/or otherwise used as a form of carbonand/or energy. For example, methane-consumption means includes, but isnot limited to, methane-utilizing microorganisms, fuel cells, turbines,reverse-flow reactors, engines, microturbines, and/or any other mode ofusing and/or consuming methane. Accordingly, in some embodiments,methane emissions are conveyed from a source to one or more of fuelcells, turbines, reverse-flow reactors, engines, or microturbines toproduce fuel or other energy. Thus, in some embodiments, methanotrophicmicroorganisms need not be used.

In one embodiment, the ammonia contained within the air is contactedwith liquid water and converted into ammonium and used as a source ofnitrogen by the methane-utilizing microorganisms. In one embodiment, thedust and/or other airborne material within the air is reduced prior toor in the course of using the methane within the air as a source ofcarbon and/or energy.

In one embodiment, methane from a first source is used by themethane-consumption means in conjunction with one or more supplementalsources of methane, such as coal mine methane, landfill gas methane,natural gas methane, manure digester methane, wastewater treatmentmethane, and/or other sources of methane.

In one embodiment, a conveyor is provided to direct, move, and/orotherwise convey methane containing air or a methane containing gaseousemission, wherein the conveyor can be used to contact the methane withthe methane consumption means. In another embodiment, a conveyer is usedto move gaseous and/or methane emissions from one location to another,and may include one or more of pipes, tubing, one or more containmentareas or compartments, ducts, channels, ventilation air or gas movingdevices (e.g., fans, vacuums, etc.), and other conduits. In oneembodiment, the conveyer is large and/or compartmentalized such that atleast a portion of the conveyer serves as a bioreactor, in that itcontains methanotrophic organisms.

In one embodiment, the methane emissions provided to the methanotrophicorganisms or other methane consumption means is provided in conjunctionwith air, dust, methane, ammonia, gases, insects, particulate matter,and/or other airborne matter. In some embodiments, one of skill in theart will appreciate that one or more of the above steps described hereinis modified or omitted. Further, the steps need not be conducted in theorder set forth herein.

DETAILED DESCRIPTION

While this invention comprises embodiments in many different forms,there will herein be described in detail preferred methods of carryingout a process (or an associated system) in accordance with severalembodiments of the invention with the understanding that the presentdisclosure is to be considered as an exemplification of the principlesof the invention and is not intended to limit the broad aspect of theinvention to the embodiments illustrated.

In a preferred embodiment of the invention, methane emissions aretreated through the use of a product-generating methanotrophic growthsystem. In one embodiment, this growth system is designed to enable theproduction of harvestable bio-based goods. For example, in a preferredembodiment, methanotrophic microorganisms and air containing methaneemissions are mutually-exposed to one another in an apparatus, such as abioreactor, filled with methanotrophic bacteria, whereby methanotrophicbacteria use methane emissions for the creation of a harvestablebio-based product. In several embodiments, the processes and systemsdisclosed herein are advantageous in that the harvestable productsgenerated are consistent over time (e.g., the functional properties of aproduct (or products) is predictable and substantially uniform frombatch to batch).

In one embodiment, the harvestable bio-based product includes, but isnot limited to, a polymer such as polyhydroxybutyrate (PHB), single cellprotein, enzymes, homogenized biomass, methanotrophic cultures, andother harvestable methanotrophic products (e.g., a harvested enzyme,such as methane monooxygenase), or combinations thereof. For example,the methods disclosed herein allow a final product that has consistentcharacteristics despite the dynamic nature of a microorganism cultureover time (e.g., the culture may undergo changes in population density,dominance of the culture by one or more species of microorganism withinthe culture, etc.). The dynamic nature of a microorganism culture, inthe absence of the methods disclosed herein, leads to a dynamic (e.g.,inconsistent) product. Such product inconsistency is associated withpoor overall quality control and potentially reduced product value, aswell as commercial non-viability. In several embodiments, the methodsdisclosed herein reduce the variability of one or more of the productsproduced by a microorganism culture.

In several embodiments, the functional properties of the products areconsistent over time. As used herein, the terms “functional properties”and “functional characteristics” shall be given their ordinary meaningsand shall also refer to the specification, features, qualities, traits,or attributes of the product. For example, in several embodiments,polymers are generated as a product. The functional characteristics ofthe generated polymers include, but are not limited to molecular weight,polydispersity and/or polydispersity index, melt flow and/or melt index,monomer composition, co-polymer structure, melt index, non-PHA materialconcentration, purity, impact strength, density, specific viscosity,viscosity resistance, acid resistance, mechanical shear strength,flexular modulus, elongation at break, freeze-thaw stability, processingconditions tolerance, shelf-life/stability, hygroscopicity, and color.As used herein, the term “polydispersity index” (or PDI), shall be givenits ordinary meaning and shall be considered a measure of thedistribution of molecular mass of a given polymer sample (calculated asthe weight average molecular weight divided by the number averagemolecular weight). Advantageously, several embodiments of the processesdisclosed herein may be carried out in sterile, semi-sterile, ornon-sterile conditions.

In several embodiments, the processes and systems disclosed herein areoptimized to produce harvestable products that have similar functionalproperties. In some embodiments, “similar” or “substantially similar”properties are those that differ by less than about 75% to about 65%,less than about 65% to about 55%, less than about 55% to about 45%, lessthan about 45% to about 35%, less than about 35% to about 25%, less thanabout 25% to about 20%, less than about 20% to about 15%, less thanabout 15% to about 10%, less than about 10% to about 5%, less than about5% to about 1%, less than about 1% to about 0.1% from batch to batch,culture to culture, sample to sample, or moment to moment. In severalembodiments, the functional properties of the products is substantiallysimilar over time, e.g., essentially indistinguishable from batch tobatch, culture to culture, or moment to moment.

The term “harvestable” as used herein shall be given its ordinarymeaning and shall also mean usable, producible, collectable, useful,yieldable, consistent, defined, commercially useful, functional, andcapable of being harvested. Likewise the term “harvest” is a broad termthat shall be given its ordinary meaning and shall also mean gather,collect, amass, accumulate, assemble, purify, isolate, use, etc.

In one embodiment, methane emissions are captured, exposed to, andtreated with one or more species of methanotrophic microorganisms toproduce a harvestable single cell protein. Single cell protein (SCP)includes microbial biomass or proteins containing therein or extractedtherefrom, and may be used as animal feed, for human nutrition, or forindustrial uses. One particularly suitable method for the production ofsingle cell protein is the use of a self-containing conglomerate ofmicroorganisms that promotes product and species stability innon-sterile or semi-sterile conditions. The production process used byNorferm A/S in Norway to create SCP from methane is one example of amethanotrophic growth process that may be applied to carry out oneembodiment of the present invention.

Another suitable method for the production of a harvestable product(including, but not limited to SCP) is the use of methods disclosedherein to promote product stability (e.g., consistency over time in theface of changing conditions in the microorganism culture) andharvestability. These methods include, but are not limited to: airdisinfection, water disinfection, mineral media disinfection, systemsterility management, directed species symbiosis, growth conditionsmanagement (e.g., manipulation or changing the formulation of the growthculture media, or other factors that influence the culturingenvironment), incoming air gaseous components separation, and others.Accordingly, in one embodiment, product stability and/or harvestabilityis enhanced or facilitated by one or more of these methods. For example,in several embodiments, the culture media which is initially used toculture methanotrophic microorganism may later be altered (e.g.,concentrations of one or more constituents increased, decreased, removedor newly introduced) in order to induce the culture to respond in acertain, uniform (e.g., across the majority of the culture) fashion, andthereby produce a desired product.

For example, in several embodiments, methane emissions are used toeffect the growth of microorganisms, wherein microorganisms aresubsequently manipulated to produce harvestable PHB by controlling theconcentration of a particular nutrient, nutrients, or combinationsthereof, such as nitrogen, magnesium, phosphorus, oxygen, carbon,potassium, sulfate and/or iron, in the culture on a batch, semi-batch,or continuous basis. As discussed above, as the microorganisms aredependent on the nutrients (including elements and other compositions)provided in a growth culture media, each component of the media can beconsidered an essential nutrient. As such, the manipulation or controlof (which includes increasing the concentration of, decreasing theconcentration of, depleting the media of such, or newly introducingsuch) one or more essential nutrient is used in several embodiments tocause a culture to metabolically respond in a known and consistentmanner, thereby ensuring predictable and consistent product generation.In several embodiments, temporal aspects of a how a microorganismculture is treated are important. For example, a particular nutrient maybe present in a growth culture medium at the outset of culturing, whenmaintenance of the culture is the primary goal. At a later time,alteration of the concentration of that particular nutrient (alone or incombination with alterations of other nutrients) is used to covert theculture from a simple growth culture to a culture producing a desiredproduct. Methanotrophic microorganisms (such as Methylocystis parvus orAlcaligenes eutrophus) generate or employ a polymer (such as PHB) as aform of an energy storage molecule to be metabolized when other commonenergy sources are not available. As is well known in the art ofmicrobial PHA and PHB production, the depletion of an essential nutrientsuch as nitrogen (or other nutrient, essential nutrient, element, orcompound present in a growth culture media) in the presence of asufficient carbon supply will cause bacterial cultures to store energyin the form of PHA, PHB, or, depending on growth conditions, somesimilar energy storage material, with the aim of accessing this storedenergy once all essential growth and reproduction components are fullypresent at a later time. Thus, in one embodiment, methanotrophicorganisms are periodically or continuously exposed to methane emissionsin a nutrient (e.g., nitrogen)-poor environment to effect PHAproduction. Partial, substantial, or complete depletion of nitrogen (orother nutrient, such as magnesium, phosphorus, potassium, zinc, sulfate,oxygen) occurs before the organisms are exposed to methane in someembodiments, or in other embodiments, after such exposure has occurred,in order to effect PHA production. Alternatively, nutrient (e.g.,nitrogen, magnesium, phosphorus, potassium, zinc, sulfate, oxygen, orother nutrient) depletion can occur at some point during exposure of theorganisms to methane in order to effect PHA production. PHB, or similarenergy storage materials, such as polyhydroxybutyrate (PHB),polyhydroxybutyrate-covalerate (PHBV), poly-4-hydroxybutyrate (P4HB),polyhydroxyhexanoate (PHHx), and polyhydroxyoctanoate (PHO), or otherPHAs, may account for a significant percentage of the weight and/orvolume of a single microorganism cell, and may be harvested by anynumber of well known techniques, such as centrifugation, cell lysis,homogenization, chloroform dissolution, sodium hydroxide dissolution,cell parts extraction, and so on.

In another embodiment of the invention, methanotrophic microorganismsare used to oxidize a quantifiable, monitored, and certifiable volume ofmethane in a sterile or non-sterile environment, including at aspecified rate, thereby creating a greenhouse gas reduction productwhich may be “harvested” and sold in a market which purchases and/ortrades greenhouse gas reduction credits, such as a carbon dioxide orcarbon dioxide equivalent credit trading market. Thus, in oneembodiment, the harvestable product is the quantifiable reduction ofmethane, especially as it pertains to air pollution reductions creditsand/or global warming gas emissions reductions credits. Accordingly, inone embodiment of the invention, a system to quantify how much methanehas been used is provided. Such embodiments are particularlyadvantageous for those organizations that need to comply with certainenvironmental regulations or need to certify that specific volumes ofmethane have been biologically oxidized.

In an additional embodiment of the invention, methane emissions may beused to create harvestable enzymes, either alone or in conjunction withthe other harvestable products disclosed herein. In several embodiments,the enzyme is methane monooxygenase. In some embodiments, the methanemonooxygenase is in a particulate form, while in some embodiments, it isin a soluble form. In one embodiment, the cellular contents ofmethanotrophic microorganisms is accessed physically, chemically,enzymatically, or otherwise to enable harvesting cell contents fromdefined (or, optionally, non-defined) microbial cultures. By way ofexample, controlling the concentration of copper (e.g., increasing,decreasing, or maintaining) in the growth culture media within certainranges of concentrations is useful, in several embodiments, to effectthe consistent production of either soluble or particulate methanemonooxygenase, as is well known in the art. In particular, in someembodiments, if the concentration of copper in a methanotrophic growthmedium is minimized and kept below specific concentrations, such as5×10⁻⁹ M, the production of soluble methane monooxygenase may beeffected in most, substantially all, or all methanotrophic cellsaccessing that copper-limited medium. In some embodiments, copper andoptionally at least one or more additional nutrient are maintained atspecific concentrations in order to effect a consistent ratio of sMMOand pMMO in a culture of methanotrophic microorganisms. In someembodiments, the production of pMMO may be effected in most or all ofthe methanotrophic cells and the production of sMMO may be substantiallyeliminated in most or all of the methanotrophic cells. In other words, avaried and dynamic culture of methanotrophic microorganisms (e.g., indifferent stages of growth or employing different active metabolicpathways) can, in some embodiments, be rendered more metabolicallyconsistent (e.g., the majority of the culture is induced to metabolizemethane through, for example, particulate methane monooxygenase) bymanipulating the concentrations of copper (and, optionally, othernutrients, including nutrients or compounds/substances that chelatecopper and thereby render them non-available to microorganisms) in themedia. In some embodiments, the methane monooxygenase is the desiredharvestable product. Soluble or particulate methane monooxygenase may beharvested using any well known methane monooxygenase extraction andpurification method.

In some embodiments, sMMO is expressed in a range between about 0% and100% of a methanotrophic culture by dry cell weight, as a percentage ofmicroorganisms expressing sMMO, or as a percentage of total MMOexpressed by one or more methanotrophic cells, including between 0% and1%, between about 1% and about 2%, between about 2% and about 3%,between about 3% and about 5%, between about 5% and about 10%, betweenabout 10% and about 20%, between about 20% and about 30%, between about30% and about 50%, between about 50% and about 70%, between about 70%and about 80%, between about 80% and about 90%, between about 90% andabout 95%, between about 95% and about or 100%, and overlapping rangesthereof. Simultaneously, or independently, in some embodiments, pMMO isexpressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingpMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout or 100%, and overlapping ranges thereof. In some embodiments, theratio of sMMO to pMMO produced in a methanotrophic culture is controlledto control the specification of PHA produced by a culture. In someembodiments, the relative weight ratio of sMMO to pMMO in amethanotrophic culture is at least or approximately 0 to 1,approximately 0.0000001 to 1, approximately 0.0001 to 1, approximately0.001 to 1, approximately 0.01 to 1, approximately 0.1 to 1,approximately 1 to 1, approximately 2 to 1, approximately 3 to 1,approximately 5 to 1, approximately 10 to 1, approximately 15 to 1,approximately 20 to 1, approximately 25 to 1, approximately 30 to 1,approximately 35 to 1, approximately 50 to 1, approximately 65 to 1,approximately 70 to 1, approximately 80 to 1, approximately 90 to 1,approximately 95 to 1, approximately 98 to 1, approximately 99 to 1,approximately 100 to 1, approximately 1000 to 1, approximately 10,000 to1, approximately 100,000 to 1, or approximately 1,000,000 to 1,respectively. In some embodiments, the relative weight ratio of pMMO tosMMO in a methanotrophic culture is approximately 0 to 1, approximately0.0000001 to 1, approximately 0.0001 to 1, approximately 0.001 to 1,approximately 0.01 to 1, approximately 0.1 to 1, approximately 1 to 1,approximately 2 to 1, approximately 3 to 1, approximately 5 to 1,approximately 10 to 1, approximately 15 to 1, approximately 20 to 1,approximately 25 to 1, approximately 30 to 1, approximately 35 to 1,approximately 50 to 1, approximately 65 to 1, approximately 70 to 1,approximately 80 to 1, approximately 90 to 1, approximately 95 to 1,approximately 98 to 1, approximately 99 to 1, approximately 100 to 1,approximately 1000 to 1, approximately 10,000 to 1, approximately100,000 to 1, or approximately 1,000,000 to 1.

In some embodiments, by controlling the relative concentrations of sMMOand pMMO produced by a culture of methanotrophic microorganisms, it ispossible to control the metabolic status of the culture and therebycontrol the type of PHA and other cellular material produced by theculture, particularly in the presence of one or more of the following:volatile organic compounds, fatty acids, volatile fatty acids, PHAs,hydroxyalkanoates, butyrate, hydroxybutyrate, polyhydroxybutyrate,valerate, hyroxyvalerate, valeric acid, butyric acid,polyhydroxybutyrate-covalerate, hexanol, heptanol, lauric acid,methanol, formate, formaldehyde, propane, ethane, butane, isobutane,acetone, acetate, acetic acid, formic acid, dissolved carbon dioxide,dissolved methane, dissolved oxygen, carbon-containing materials,ammonia, ammonium, and other elements or compounds or substances thatimpact the metabolism of a culture of methanotrophic microorganisms in acertain manner, including according to the relative concentration ofsMMO or pMMO in such a culture. In some embodiments, sMMO and/or pMMO isexpressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingsMMO or pMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout or 100%, and overlapping ranges thereof prior to, during,throughout, or after a PHA production phase.

In one embodiment, sMMO is not expressed, or is expressed in lowconcentrations (e.g., less than 5%, 3% or 1%), in a methanotrophicculture prior to, during, throughout, or after a PHA production phase.In some embodiments, the directed or controlled absence or reduction ofsMMO in a methanotrophic culture producing PHA, particularly in thepresence of non-methane organic compounds or substances that can bemetabolized by methanotrophic microorganisms, engenders PHA productionstability, consistency, and control by selectively shielding against themetabolism of one or some or many non-methane organic compounds orsubstances that might otherwise be metabolized in the presence of sMMO,which enables the metabolism of a larger group of non-methane compoundsor substances than pMMO. Further, in some methanotrophic cultures andsome embodiments of the invention, pMMO promotes PHA synthesis at highintracellular concentrations by reducing cellular production of non-PHAmaterials, particularly as compared to PHA synthesis using sMMO.Similarly, in one embodiment, pMMO is not expressed, or is expressed inlow concentrations (e.g., less than 5%, 3% or 1%), in a methanotrophicculture prior to, during, throughout, or after a PHA production phase.In some embodiments, the directed or controlled absence or reduction ofpMMO in a methanotrophic culture producing PHA, particularly in thepresence of non-methane compounds or substances (organic or inorganic)that can be metabolized by methanotrophic microorganisms, engenders PHAproduction stability, consistency, and control by selectively inducingor promoting the metabolism of one or some or many non-methane organiccompounds that might otherwise be not be metabolized using pMMO.Further, in some methanotrophic cultures, sMMO promotes PHA synthesis athigh intracellular concentrations by reducing cellular production ofnon-PHA materials, particularly as compared to PHA synthesis using pMMO.By controlling the concentration of sMMO relative to pMMO in amethanotrophic microorganism culture in the presence of methane and/ornon-methane organic or inorganic compounds, including VOCs, volatilefatty acids, methanol, formaldehyde, acetone, formate, ethane, propane,alkanoic acids, or carbon dioxide, it is possible to control thespecification or type of PHA produced by the culture, including themolecular weight, polydispersity, and other similar functionalcharacteristics. In some embodiments, it is preferable to maintain theconcentration of copper in the culture media in order to promote sMMOproduction. In some embodiments, the production of sMMO in many, most,or substantially all of the methanotrophic cells enables the culture toproduce more PHA when subject to a nutrient limiting step than wouldotherwise be produced if the relative ratio of pMMO in the culture washigher prior to the nutrient limiting step. In some embodiments, it ispreferable to maintain the concentration of copper in the culture mediain order to promote pMMO production. In some embodiments, the productionof pMMO in many, most, or substantially all of the methanotrophic cellsenables the culture to produce more PHA when subject to a nutrientlimiting step than would otherwise be produced if the relative ratio ofsMMO in the culture was higher prior to the nutrient limiting step. Inone embodiment, one or more methanotrophic cells or cultures are subjectto repeated growth and PHA synthesis cycles or steps, wherein theproduction of methane monooxygenase is followed by the production ofPHA, wherein such cycling order is repeated over at least twoconsecutive cycles, and wherein the relative concentration of sMMO topMMO in the cells or cultures is controlled or caused to remainapproximately similar (e.g., within about 5% to about 10%, with about10% to about 20%, within about 20% to about 30%, within about 30% toabout 40%, within about 40% to about 50%, within about 50 to about 75%)or the same in each new cycle or step in order to control the functionalproperties of the PHA produced by or extractable from the culture orcultures in each new or repetitive cycle with the same or new cells.

In certain embodiments, as discussed above, the control of theconcentration of one or more essential nutrients results in theproduction of a desired type of methane monooxygenase. In someembodiments, the production of the desired type of methane monooxygenaseis followed by the controlling (e.g., increase, decrease, ormaintenance) of an essential nutrient that results in the induction ofPHA production. In some embodiments, these two steps performedconsecutively can be considered a production cycle. In some embodiments,the first essential nutrient is the same as the second essentialnutrient. In some embodiments, the concentration is controlled in thesame fashion between the steps of the production cycle, but to differentdegrees (e.g., reduced by 20% in the first step and by a further 20% inthe second step). In other embodiments, the concentration is controlledin different fashion (e.g., increased in one step and decreased in theother) to the same or varying degrees. As used herein, the term“production cycle” shall be given its ordinary meaning, and shall alsorefer to the sequential steps that result in the production of PHAhaving consistent functional properties. In some embodiments of themethods disclosed herein a single production cycle is used, whileanother by embodiments, a plurality of production cycles is performed.In some embodiments, additional steps are included in the productioncycle. In some embodiments the repetition of production cycles improvesthe overall PHA (or other product) quality (e.g., purity, functionalperformance) and/or output (e.g., rate or yield of production) for agiven amount of input material (methane emissions or othermethane-containing gas), including by selecting for, controlling, and/orenhancing the metabolic disposition of the microorganism culture toproduce PHA at higher quality, rates, or yield for a given inputmaterial.

In several embodiments of the invention, a culture is induced tomaximize the production and intracellular concentration of PHA in theculture by controlling the concentrations of essential nutrients and/orchemicals in the medium and causing the culture to expel non-PHA biomassmaterial, including water-soluble material, into the medium, therebyincreasing the concentration of PHA in the culture to greater than 70%PHA, greater, than 80% PHA, greater than 85% PHA, greater than 90% PHA,greater than 95% PHA, or greater than 99% PHA. In another embodiment ofthe invention, the culture is induced to maximize the production andintracellular concentration of PHA in the culture by controlling theconcentrations of essential nutrients and/or chemicals in the medium andcausing the culture to synthesize PHA to very high concentrations,increasing the concentration of PHA in said culture to greater than 70%PHA, greater, than 80% PHA, greater than 85% PHA, greater than 90% PHA,greater than 95% PHA, or greater than 99% PHA. In one embodiment,PHA-biomass may or may not be subjected to one or more extractiontechniques in various degrees, steps, or combinations, such as solventextraction, super critical carbon dioxide extraction, non-polymercellular material dissolution extraction, or other extractiontechniques. In one embodiment, non-PHA biomass material expelled intothe mineral medium is separated from the PHA-rich culture material byone or more separation mechanisms, including, but not limited toliquid-liquid and liquid-solid separation (e.g., filtration,centrifugation, reverse osmosis, ultrafiltration, distillation, etc.).In one embodiment of the invention, the culture is induced to solubilizenon-PHA material into the medium by controlling the concentrations ofone or more essential nutrients and/or chemicals in the medium. In oneembodiment, solubilized non-PHA biomass material is separated from PHAby various separation mechanisms, including liquid-liquid andliquid-solid separation (e.g., filtration, centrifugation, distillation,etc.). In one embodiment, the culture is induced to increase theconcentration of PHA as a percentage of solid material in the medium bycontrolling the concentrations of one or more essential nutrients orchemicals in the medium, thereby causing the culture to a) expel non-PHAmaterial into the mineral medium, b) solubilize non-PHA material intowater-soluble material, c) expel water-soluble non-PHA material into themedium, and/or d) dissolve non-PHA material into the medium.

Likewise, in some embodiments, adjusting the growth culture medium withrespect to other compounds, substances, or nutrients can impact thecultured microorganism in other beneficial ways (e.g., to induceproduction of another or an alternative product). As discussed above, inseveral embodiments, the concentration of one or more essentialnutrients is adjusted (e.g., increased, decreased, or depleted) in themedia, which, in several embodiments, causes the microorganisms to storeenergy in the form of a polymer, which can thereafter be harvested. Insome embodiments, the manipulation of the culture environment in whichall the microorganisms of a given culture are growing or maintaining anactive or responsive metabolic state allows a large portion (if not all)of the microorganisms in the culture to respond to the manipulation in auniform fashion (e.g., most or all store energy as PHA), which leads tomore uniform products, due, at least in part, to the uniformity of themicroorganism response. In some embodiments, between about 30% and about50%, between about 50% and about 70%, between about 70% and about 80%,between about 80% and about 90%, between about 90% and about 95%,between about 95% and about or 100% (and overlapping ranges thereof) ofthe culture responds to a change (or maintenance) of certain cultureconditions in the same (or a substantially similar) fashion.

The processes disclosed herein may be carried out and directed in acontrolled bioreactor, wherein liquid, semi-liquid, particulate, orsolid mineral media may be used to enhance the growth of methanotrophicmicroorganisms. Alternatively, the processes described herein may becarried out in reaction tanks, vessels, fixed film reactors, trickle bedreactors, foam reactors, or any other appropriate culture/containmentsystems.

In additional embodiments, various processing techniques known in theart may or may not be used to preferentially extract (e.g., removebiomass or biocatalyst from) harvestable products of methanotrophicgrowth, such as chemical treatment, centrifugation, drying, andhomogenization. In some embodiments, extraction agents or mechanisms areselected from the group consisting of: methylene chloride, acetone,ethanol, methanol, dichloroethane, supercritical carbon dioxide,sonication, homogenization, water, heat, distillation, spray drying,freeze drying, centrifugation, filtration, enzymes, surfactants,hydrolyzers, acids, bases, hypochlorite, peroxides, bleaches, ozone,EDTA, and/or combinations thereof.

In one embodiment, the extraction process may be substantially carriedout at intracellular temperatures less than 100° C. In otherembodiments, temperatures for extraction range from about 10° C. toabout 30° C., from about 30° C. to about 50° C., from about 50° C. toabout 70° C., from about 70° C. to about 90° C., from about 90° C. toabout 120° C., from about 100° C. to about 140° C., from about 20° C. toabout 150° C., or from about 120° C. to about 200° C., or higher. Inanother embodiment, cells and/or biocatalyst are reused forpolymerization following the extraction process as viable cells orcatalytic material.

In a several preferred embodiments of the invention, landfill gas isused as the source of methane. In one embodiment, impurities fromlandfill gas, such as non-methane and/or volatile organic compounds,water vapor, and/or carbon dioxide are partially, substantially, orcompletely removed. In another embodiment, the landfill gas isdisinfected. In one embodiment, UV treatment is used to disinfect thegas. Mechanical, activated carbon, or chemical filtration may also beused. However, in several embodiments the landfill gas is used withoutpurification, disinfection, or other such manipulation.

In several embodiments, methane emissions within landfill gas (or othersource of methane) are exposed to methanotrophic microorganisms. In oneembodiment, gaseous emissions comprising methane are fed into abioreactor containing methanotrophic microorganisms suspended in or on aliquid, semi-liquid, or solid growth-culture medium containing growthmedia comprising essential nutrients. In another embodiment, aftermethanotrophic microorganisms have grown and reproduced using methaneemissions as a source of carbon and/or energy, these microorganisms areharvested as single cell protein through various extraction andde-watering processes. In some embodiments, non-methane componentswithin methane emissions, such as methanol, acetone, acetate, formate,formaldehyde, hydroxyalkanoates, hydroxybutyrate, octanoic acid,octanol, carbon dioxide, nitrogen, oxygen, di-oxygen, di-nitrogen,water, water vapor, argon, ethane, propane, butyrate, butyric acid,hexanoic acid, hexanol, heptanoic acid, heptanol, pentane, pentanoicacid, and volatile organic compounds, are used to modify the functionaland/or molecular characteristics of PHA produced by a microorganismculture, e.g., causing methanotrophic microorganisms to produce varioustypes of PHAs, such as polyhydroxybutyrate (PHB), high ultra highmolecular weight PHB, polyhydroxybutyrate-covalerate (PHBV),poly-4-hydroxybutyrate (P4HB), polyhydroxyhexanoate (PHHx), andpolyhydroxyoctanoate (PHO), or other PHAs with enhanced or modifiedproperties according to the kinds of non-methane components exposed tothe methanotrophic microorganisms.

In several embodiments, a method of treating gaseous emissions (e.g.,landfill gas) is provided. In one embodiment, the method comprises: (i)enclosing the landfill gas in a bioreactor containing methanotrophicmicroorganisms; and (ii) harvesting the microorganisms and/or theproducts produced by the microorganisms in the bioreactor. In anotherembodiment, the method comprises: (i) removing impurities from thelandfill gas; (ii) disinfecting the landfill gas; (iii) enclosing thelandfill gas in a bioreactor containing methanotrophic microorganisms;and (iv) harvesting the microorganisms and/or the products produced bythe microorganisms in the bioreactor.

In several embodiments, a portion of the microorganisms comprising sMMOand/or pMMO are directed into a bioreactor, or another phase ofbioreactor operation, containing or comprising a nitrogen depletedgrowth medium (or a medium deprived of one or more other essentialnutrients) and a constant supply of gaseous emissions (e.g., landfillgas), whereby the microorganisms synthesize intracellular PHA (e.g.,PHB). In one embodiment, the PHB-filled cells are subsequently removedfrom the reactor in order to process and harvest the material intocommercially useful form. These processes are preferentially carried outon a continuous, semi-continuous, semi-batch, or batch-wise basis, usingmethane emissions from any source, including, but not limited tolandfills, coal mines, wastewater treatment plants, agriculturalsystems, petroleum systems, or other sources.

As used herein, the term “methanotrophic microorganisms” shall be givenits ordinary meaning and shall also refer to any microorganisms thatutilize methane as a source of carbon and/or energy for growth andreproduction, including naturally-occurring and/or geneticallyengineered microorganisms. It shall also refer to the combination ormixture of methanotrophic and non-methanotrophic microorganisms thatpromote the growth of methanotrophic microorganisms. In one preferredembodiment, this combination comprises Methylococcus capsulatus,Alcaligenes acidovorans, Bacillus firmus, and Bacillus brevis, sincethis combination has been shown to limit or reduce bacterialcontamination in non- and semi-sterile bioreactor conditions, therebyenabling stable product formation. In another preferred embodiment, thiscombination comprises any methanotrophic microorganisms, such as Type IImethanotrophic microorganisms, including from the genus Methylocystisand Methylosinus that may be preferentially used to produce polymerssuch as PHB, enzymes such as methane monooxygenase, and/or any othercellular components. In another preferred embodiment, this combinationcomprises a non-defined mix of methanotrophic and non-methanotrophicmicroorganisms that can be used to create a harvestable product from theoxidation (or alternate processing) of methane emissions.

The terms “methanotrophic microorganism growth-culture medium” and“growth medium” shall be given their ordinary meaning and shall alsorefer to any medium promoting the growth of microorganisms. The termsshall also refer to any substrate, aside from methane, whichmicroorganisms oxidize or otherwise break down. It shall also refer toany substrate or material that concentrates methane, preferentiallysequesters methane, “traps” methane, increases the solubility and/oravailability of methane, and/or otherwise enables the enhancedbreakdown, oxidation, and/or utilization of methane by microorganisms.The term “microorganism growth-culture medium” includes, but is notlimited to, any substrate and/or microorganism immobilization means,such as liquid, semi-liquid, gas, particulate, ceramic, foam, plastic,alginate gel, fixed film, attached biofilm, methanol-enriched,copper-enriched, clay, nutrient, or other appropriate growth-culturemedium. In one preferred embodiment, this growth culture mediumcomprises an aqueous solution containing water, nitrogen, ammonium,trace minerals, and other well-known microorganism growth-culture mediumcomponents necessary for the growth and reproduction ofmethane-utilizing bacteria (e.g., essential nutrients), such as, forexample, carbon, oxygen, magnesium, phosphorus, phosphate, potassium,sulfate, sulfur, calcium, boron, aluminum, chromium, cobalt, iron,copper, nickel, manganese, molybdenum, sodium, nitrate, ammonia,ammonium, urea, amino acids, methane, carbon dioxide, and/or hydrogen.In another preferred embodiment, this growth culture medium comprises amicroorganism immobilization means, such as organic or inorganicparticles, on which a liquid or semi-liquid mineral medium solution iscontinuously or periodically contacted and on which microorganisms areattached. In another preferred embodiment, this growth-culture mediumcomprises waste organic materials, which methane-utilizingmicroorganisms may or may not break down to produce a byproduct oforganic materials that may or may not be useful. In another preferredembodiment, this growth-culture medium comprises a liquid foamsubstrate.

As is well-known in the art, the various components of a growth-culturemedium consist of those compounds, substances, salts, elements, andother nutrients that are essential for the continued viability andgrowth of methanotrophic microorganisms.

In another preferred embodiment, a system comprising methanotrophicorganisms is used to degrade or otherwise reduce a pollutant other thanmethane as a method to enable the viable treatment of methane emissions.In one embodiment, the growth of methanotrophic microorganisms usingmethane emissions is carried out in the presence of a non-methanematerial that can be broken-down, oxidized, consumed, and/or otherwisechanged in form through the action of such microorganisms. In severalembodiments, the non-methane material includes, but is not limited to,one or more of the following: toluene, benzene, methanol, propylene, anyalkenes, alcohols, ethers, alicyclics, aromatics, and/or chlorinatedorganic compounds, such as the pollutant TCE. As discussed herein, theresultant product(s), including the oxidized chemical or quantifiablepollutant treatment, may be harvested in a controlled, directed, and/orquantifiable manner.

In another preferred embodiment of the invention, following the growthof methanotrophic microorganisms in a bioreactor (or other appropriateapparatus), some or all of the contents of the bioreactor are removedfrom the bioreactor and are either processed or used and sold directly.Processing may include any number of methods that enable productharvest, such as centrifugation, filtration, drying, homogenization,chemical treatment, physical treatment, enzymatic treatment, or anyother processing means. Processing means may be used to extract productsout of defined or non-defined conglomerates of methanotrophicmicroorganisms. The application and utilization of such processingtechniques, such as, for example, centrifugation and homogenization, maybe used to effect the overall harvestability of the methanotrophicgrowth and treatment process, especially where the maintenance of adefined culture is unfeasible or otherwise impractical. For example, ifa methanotrophic culture is particularly varied (e.g., a large number ofspecies) or dynamic over time due to changing culture conditions, aportion of the culture can be removed at a first point in time,processed to obtain a desired product and stored. Thereafter, asubsequent portion of the culture can be removed at a time when thedesired product is again being produced by the culture, and optionallycombined with the first batch of product. In this manner a moreconsistent product over time can be obtained from a varied culturethrough application of the various processing means disclosed herein.

Preferred embodiments of the present invention offer one or moreadvantages. For example, one or more embodiments provide one or more ofthe following benefits:

(i) enables the viable and economical treatment of methane emissions;

(ii) enables the viable and economical application of methanotrophicmicroorganisms to methane emissions treatment in environments,particularly for methane emissions streams where the concentration ofmethane is low, variable, impure, or unpredictable;

(iii) provides a methanotrophic methane emissions treatment process thatis economically competitive with alternative methods of methaneemissions treatment;

(iv) overcomes previously insurmountable practical challenges in thefield of methane emissions treatment, particularly for low-quality, lowpurity, or low-BTU methane emissions treatment; and/or

(v) provides a process which, if widely applied, has the capacity tosignificantly reduce global methane emissions.

Preferred embodiments of the invention comprise one or more of theforegoing advantages and/or objects. Further objects and advantages willbecome apparent from the ensuing description.

In several embodiments, methane emissions may be used from a variety ofsources or combinations of sources, including, but not limited tolandfills, coal mines, wastewater treatment plants, manure digesters,agricultural digesters, compost heaps, enclosed agricultural feedlots,leaking or otherwise emitting petroleum systems, and any other source ofmethane emissions or off-gas whereby the creation of harvestablebio-based is enabled. The methane emitted by ruminant animals can alsobe used as a source of methane according to several embodiments. Theprocessing of methane emissions produced by ruminant animals isdiscussed in greater details in U.S. Pat. No. 7,745,197, which isincorporated in its entirety by reference herein.

The term “consolidation means” shall be given its ordinary meaning andshall also refer to any means by which methane emissions are unified,mutually-directed, and/or otherwise consolidated for conveyance,movement, or storage. In one preferred embodiment, a consolidation meanscomprises an air-tight ducting tube running from an air outlet to amutual-exposure means, as described below, wherein methane containinggas is directed from a first location, through a consolidation means,and into a mutual-exposure means. In another preferred embodiment, aconsolidation means comprises multiple ducting tubes connected to airoutlets and situated to consolidate methane containing gas into a singleducting tube that ultimately leads into a methane-consumption system.

The term “ventilation means” shall be given its ordinary meaning andshall also refer to any means by which air, gases, and/or other airbornematerial is mechanically forced, pushed, pulled, drawn, moved, conveyed,or otherwise directed into, through, and/or out of a first area (e.g., asource of methane containing gas) to a second area (e.g., a bioreactor).

The term “air inlet” shall be given its ordinary meaning and shall alsorefer to any location where air, gas, and/or other airborne materialenters into an area or chamber that is fully or partially enclosed(e.g., a bioreactor).

The term “air outlet” shall be given its ordinary meaning and shall alsorefer to any location where air, gas, and/or other airborne materialexits out an area or chamber that is fully or partially enclosed (e.g.,a bioreactor).

Methane-utilizing microorganisms represent one embodiment of a“methane-consumption system” or “methane consumption means.” The lattertwo terms shall be given their ordinary meaning and shall also refer toone or more biological systems that utilize enteric fermentation methaneas a source of carbon and/or energy, a mechanical system that uses orconsumes methane, and/or a chemical system that uses, degrades,consumes, or reacts with methane.

The term “methane-utilizing microorganism” or “methanotrophicmicroorganism” shall be used interchangeably, shall be given theirordinary meaning, and shall also refer to any microorganism,naturally-occurring or genetically-engineered, that utilizes methane,including enteric fermentation methane, as a source of carbon and/orenergy. The term “methane-utilizing microorganisms” also refers to thecombination of methane-utilizing and non-methane-utilizingmicroorganisms that are collectively associated with the growth ofmethane-utilizing microorganisms. In one embodiment, this microorganismcombination includes one or more of the following: Methylococcuscapsulatus, Alcaligenes acidovorans, Bacillus firmus, and Bacillusbrevis. In one embodiment, a combination of these microorganisms is usedbecause among other advantages, this combination is known to promote thelong-term growth of Methylococcus capsulatus. The term“methane-utilizing microorganisms” also includes any methanotrophicorganisms and organisms that use or “take-up” methane. In severalembodiments, methane-utilizing microorganisms are confined in amicroorganism holding tank containing methane-utilizing microorganismsand a microorganism growth-culture medium. in several embodiments, abiofiltration system containing methane-utilizing microorganisms isprovided, wherein microorganisms either are or are not attached to amicroorganism support substrate and are continuously or intermittentlycontacted with a microorganism growth-culture medium. In severalembodiments, the microorganism are used in a bioreactor containing amicroorganism growth-culture medium wherein the growth-culture medium isin liquid, foam, solid, semi-solid, or any other suitable form andmethane-utilizing microorganisms are naturally-occurring and/orgenetically engineered and may or may not have been selectively insertedas part of a pre-determined microbial consortium. While the use of aspecified microorganism consortium may provide some benefits, and isused in some embodiments, in other embodiments, a non-specified andnaturally-equilibrating consortium of one or more microorganisms isemployed. Typical examples of methane-utilizing microorganisms useful inseveral embodiments of the present invention include, but are notlimited to, bacteria and yeast.

Suitable yeasts include species from the genera Candida, Hansenula,Torulopsis, Saccharomyces, Pichia, 1-Debaryomyces, Lipomyces,Cryptococcus, Nematospora, and Brettanomyces. The preferred generainclude Candida, Hansenula, Torulopsis, Pichia, and Saccharomyces.Examples of suitable species include: Candida boidinii, Candidamycoderma, Candida utilis, Candida stellatoidea, Candida robusta,Candida claussenii, Candida rugosa, Brettanomyces petrophilium,Hansenula minuta, Hansenula saturnus, Hansenula californica, Hansenulamrakii, Hansenula silvicola, Hansenula polymorpha, Hansenulawickerhamii, Hansenula capsulata, Hansenula glucozyma, Hansenulahenricii, Hansenula nonfermentans, Hansenula philodendra, Torulopsiscandida, Torulopsis bolmii, Torulopsis versatilis, Torulopsis glabrata,Torulopsis molishiana, Torulopsis nemodendra, Torulopsis nitratophila,Torulopsis pinus, Pichia farinosa, Pichia polymorpha, Pichiamembranaefaciens, Pichia pinus, Pichia pastoris, Pichia trehalophila,Saccharomyces cerevisiae, Saccharomyces fragilis, Saccharomyces rosei,Saccharomyces acidifaciens, Saccharomyces elegans, Saccharomyces rouxii,Saccharomyces lactis, and/or Saccharomyces fractum.

Suitable bacteria include species from the genera Bacillus,Mycobacterium, Actinomyces, Nocardia, Pseudomonas, Methanomonas,Protaminobacter, Methylococcus, Arthrobacter, Methylomonas,Brevibacterium, Acetobacter, Methylomonas, Brevibacterium, Acetobacter,Micrococcus, Rhodopseudomonas, Corynebacterium, Rhodopseudomonas,Microbacterium, Achromobacter, Methylobacter, Methylosinus, andMethylocystis. Preferred genera include Bacillus, Pseudomonas,Protaminobacter, Micrococcus, Arthrobacter and/or Corynebacterium.Examples of suitable species include: Bacillus subtilus, Bacilluscereus, Bacillus aureus, Bacillus acidi, Bacillus urici, Bacilluscoagulans, Bacillus mycoides, Bacillus circulans, Bacillus megaterium,Bacillus licheniformis, Pseudomonas ligustri, Pseudomonas orvilla,Pseudomonas methanica, Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas boreopolis,Pseudomonas pyocyanea, Pseudomonas methylphilus, Pseudomonas brevis,Pseudomonas acidovorans, Pseudomonas methanoloxidans, Pseudomonasaerogenes, Protaminobacter ruber, Corynebacterium simplex,Corynebacterium hydrocarbooxydans, Corynebacterium alkanum,Corynebacterium oleophilus, Corynebacterium hydrocarboclastus,Corynebacterium glutamicum, Corynebacterium viscosus, Corynebacteriumdioxydans, Corynebacterium alkanum, Micrococcus cerificans, Micrococcusrhodius, Arthrobacter rufescens, Arthrobacter parafficum, Arthrobactercitreus, Methanomonas methanica, Methanomonas methanooxidans,Methylomonas agile, Methylomonas albus, Methylomonas rubrum,Methylomonas methanolica, Mycobacterium rhodochrous, Mycobacteriumphlei, Mycobacterium brevicale, Nocardia salmonicolor, Nocardia minimus,Nocardia corallina, Nocardia butanica, Rhodopseudomonas capsulatus,Microbacterium ammoniaphilum, Archromobacter coagulans, Brevibacteriumbutanicum, Brevibacterium roseum, Brevibacterium flavum, Brevibacteriumlactofermentum, Brevibacterium paraffinolyticum, Brevibacteriumketoglutamicum, and/or Brevibacterium insectiphilium, including, but notlimited to, microorganisms that utilize the serine, ethylmalonyl-CoA,and/or ribulose monophosphate (RuMP) pathway(s).

The term “microorganism growth-culture medium” shall be given itsordinary meaning and shall also refer to any medium promoting the growthof microorganisms. It shall also refer to any substrate, aside frommethane, which microorganisms oxidize or otherwise break down. It shallalso refer to any substrate or material that concentrates methane,preferentially sequesters methane, “traps” methane, increases thesolubility and/or availability of methane, and/or otherwise enables theenhanced breakdown, oxidation, and/or utilization of methane bymicroorganisms. The term “microorganism growth-culture medium” includes,but is not limited to, any substrate and/or microorganism immobilizationmeans, such as liquid, semi-liquid, gas, particulate, ceramic, foam,plastic, alginate gel, methanol-enriched, copper-enriched, clay,nutrient, or other appropriate growth-culture medium. In one preferredembodiment, this growth culture medium comprises aqueous solutioncontaining water, nitrogen, ammonium, trace minerals, and otherwell-known microorganism growth-culture medium components necessary forthe growth and reproduction of methane-utilizing bacteria, such as, forexample, carbon, oxygen, magnesium, phosphorus, phosphate, potassium,sulfate, sulfur, calcium, boron, aluminum, chromium, cobalt, iron,copper, nickel, manganese, molybdenum, sodium, ammonia, ammonium, urea,amino acids, methane, carbon dioxide, and/or hydrogen. In anotherpreferred embodiment, this growth culture medium comprises amicroorganism immobilization means, such as organic or inorganicparticles, on which a liquid or semi-liquid mineral medium solution iscontinuously or periodically contacted and on which microorganisms areattached. In another preferred embodiment, this growth-culture mediumcomprises waste organic materials, which methane-utilizingmicroorganisms may or may not break down to produce a byproduct oforganic materials that may or may not be useful. In another preferredembodiment, this growth-culture medium comprises a liquid foamsubstrate.

In yet another preferred embodiment, the growth-culture medium iscombined with various materials which methane-utilizing microorganismsmay or may not convert to more desirable materials. Examples of variousmaterials include, but are not limited to, toluene, trichloroethylene(TCE), propylene, and agricultural byproduct materials whichmicroorganisms may preferentially breakdown or oxidize.

As discussed above, several embodiments of the methods and systemsprovided herein are advantageous in that low concentration, variableflow, unpredictable, or non-pure methane streams or emissions,previously unusable, are used for the conversion of methane into usefulproducts (e.g., polymers, proteins, enzymes, heat, and/or electricity).In one embodiment, methane is capable of being used at a methane-in-airvolumetric concentration down to abut 0.1% methane-in-air, specificallyby methanotrophic microorganisms, catalytic reactors, and thermalflow-reversal reactors. Thus, systems such as those disclosed herein canbe used, in some embodiments, as a way to utilize low-concentrationsources of methane to produce polymers, proteins, enzymes, heat,electricity, and/or other defined and consistent products. Specifically,microturbines, fuel cells, reverse-flow reactors, methanotrophicmicroorganisms and other means capable of utilizing methane at lowconcentrations can be used as a methane-consumption means in accordancewith several embodiments of the invention, allowing air containing lowconcentrations of methane to be used in an unadulterated state as viablefeedstock fuel. Optionally, gas concentrators that increasemethane-in-air concentrations of exhaust gas, including systems thatsupplement or add other sources of methane, are employed to increasemethane concentrations to levels more suitable for use by a range ofmethane-consumption means. Thus, although one preferred embodimentdetails the use of methane-utilizing microorganism as a preferredmethane-consumption means, in another embodiment, any number ofmethane-consumption means (or combinations thereof) may be employed inaccordance with embodiments of the invention to convert air containinglow concentrations of methane into useful products such as heat and/orelectricity.

The following Example illustrates non-limiting embodiments of thepresent invention and is not intended in any way to limit the claimedinvention. Moreover, the methods described in the following Example neednot be performed in the sequence presented.

EXAMPLE 1

The following example describes the processing of methane emissions froma landfill site in accordance with several embodiments disclosed herein.It shall be appreciated by one of skill in the art that the methoddescribed herein can also be used for any site that produces methane,such as coal mines, wastewater treatment plants, manure digesters,agricultural digesters, compost heaps, enclosed agricultural feedlots,fossil fuel systems, or combinations thereof.

In one embodiment, a landfill site that produces methane emissions willbe identified. Landfill gas extraction wells and/or blowers are employedto draw landfill gas out of the landfill using equipment and technologythat is used by any landfill gas extraction or environmental servicesfirm, such as LFG Technologies of Fairport, N.Y., USA or SCS Engineersof Long Beach, Calif., USA. In several embodiments, the methane contentof the extracted landfill gas can be monitored for the production ofmethane using any methane detector commonly used by an environmentalservices firm. If the methane concentration is greater than about 0.1%to about 1.0%, the landfill will be deemed suitable for methane recoveryand processing according to several embodiments disclosed herein. Insome embodiments, the methane concentration is between about 10% andabout 60%, including between about 10% and about 20%, between about 20%and 30%, between about 30% and 40%, or more preferably between about 40%and about 50%. In other embodiments, methane emissions comprise methanein a concentration in the range of about 0.1% to about 10% (includingabout 0.1% to about 1%, about 1% to about 2%, about 2% to about 3%,about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% toabout 10%, and overlapping ranges thereof) in the range of about 10% toabout 20%, or in the range of about 20% to about 40%, or greater thanabout 20%. Landfill sites (or other sites) having methane concentrationsless than about 0.1% and greater than about 60% may also be used in someembodiments of the invention.

After a suitable landfill site has been identified, the landfill gaswill be captured from the landfill using an air compressor, blower,vacuum, or other suitable capturing means. Optionally, impurities willthen be removed from the landfill gas. For example, non-methane organicor inorganic compounds or substances, such as methanol, acetone,acetate, formate, formaldehyde, hydroxyalkanoates, hydroxybutyrate,octanoic acid, octanol, carbon dioxide, nitrogen, oxygen, di-oxygen,di-nitrogen, water, water vapor, argon, ethane, propane, butyrate,butyric acid, hexanoic acid, hexanol, heptanoic acid, heptanol, pentane,pentanoic acid, and volatile organic compounds, can be removed bypassing the landfill gas through activated carbon, leaving mostlymethane and carbon dioxide as the main components of the landfill gas.Although impurities need not be removed in every embodiment of theinvention, the removal of impurities is advantageous in someembodiments. One advantage of removing impurities (such as water vapor,volatile organic compounds, particulate materials, and/or carbondioxide) is minimizing the possibility of hindering microorganism growthas microorganisms contact the landfill gas.

The landfill gas is optionally disinfected using UV light. In thoseembodiments in which impurities are removed, UV irradiation can be usedbefore, after or during the removal process. UV irradiation may also beused in embodiments that do not employ impurities removal. UV light isbelieved to disinfect the landfill gas by disrupting the nucleic acidstructures within microorganisms in the landfill gas, subsequentlyeliminating the capacity of these microorganisms to reproduce.Impurities removal and disinfection do not have to be employed, however,because methanotrophic microorganisms can and are genetically ormetabolically equipped (or can be designed to be so equipped) towithstand a range of impurities.

The landfill gas (which in a preferred embodiment is purified anddisinfected) as well as air or oxygen (which in one embodiment ispurified and/or disinfected) will be fed into a self-contained enclosureusing an air compressor, air blower, or similar means. Theself-contained enclosure is preferably a bioreactor that contains atleast one species of methanotrophic microorganisms and growth medium,wherein the growth medium contains one or more non-methane compound orsubstance such as methanol, acetone, acetate, formate, formaldehyde,hydroxyalkanoates, hydroxybutyrate, octanoic acid, octanol, carbondioxide, nitrogen, oxygen, di-oxygen, di-nitrogen, water, water vapor,argon, ethane, propane, butyrate, butyric acid, hexanoic acid, hexanol,heptanoic acid, heptanol, pentane, pentanoic acid, or volatile organiccompounds. The bioreactor is preferably sized to accommodate the flowrate of landfill gas to be treated. For example, a bioreactor treating1000 cubic feet per minute of landfill gas should be approximately twiceas large in volume as a bioreactor treating 500 cubic feet per minute oflandfill gas. Preferably, a bioreactor treating 1000 cubic per minute oflandfill gas will contain about 10,000-800,000 liters of growth mediumcontaining suspended methanotrophic microorganisms. Growth medium can bea liquid, semi-liquid, or solid substrate. For example, the growthmedium may be water containing growth nutrients such as nitrogen,magnesium, phosphorus, copper, iron, potassium, and trace minerals, inwhich microorganisms are suspended.

In one embodiment, the growth medium is tailored to meet thespecification of the end-product of microorganism growth. If thebioreactor is being used or processed, according to an embodiment of theinvention, to create soluble methane monooxygenase, for example, it willbe preferable to keep the copper concentration in the growth mediumsufficiently low, for example, below about 5×10⁻⁹ M, which may beachieved through continuous monitoring of the growth medium, calculatedmetering of copper into the growth medium, metering of copper-containingwater into the growth medium, or, e.g., calculated metering of copperchelating agent into the growth medium.

The growth medium solution may consist of water filled with a range ofmineral salts (e.g., essential nutrients, such as carbon, hydrogen,nitrogen, oxygen, phosphorus, potassium, calcium, sodium, chlorine,methane, carbon dioxide, magnesium, iron, copper, sulfate, manganese,boron, zinc, aluminum, nickel, chromium, cobalt, or molybdenum). Forexample, each liter of growth medium may be comprised of 1 g KH₂PO₄, 1 gK₂HPO₄, 1 g KNO₃, 1 g NaCl, 0.2 g MgSO₄, 26 mg CaCl₂*2H₂O, 5.2 mg EDTANa₄(H₂O)₂, 1.5 mg FeCl₂*4H₂O, 0.12 mg CoCl₂*6H₂O, 0.1 mg MnCl₂*2H₂O,0.07 mg ZnCl₂, 0.06 mg H₃BO₃, 0.025 mg NiCl₂*6H₂O, 0.025 mg NaMoO₄*2H₂O,0.015 mg CuCl₂*2H₂O, or a combination thereof. In another embodiment,the growth medium comprises solid and/or liquid media. In yet anotherembodiment, the growth medium comprises agar.

Methanotrophic microorganisms may be present in the bioreactor in anyconcentration. Preferably, in one embodiment, there are about 1 to 100grams of microorganisms per liter of water (or other aqueous solution)in the bioreactor, preferably about 10-250 grams per liter, morepreferably about 40-200 grams per liter, over the course of treatment.The methanotrophic microorganisms are exposed to the methane withinlandfill gas for about 0.1-200 hours, whereupon a portion of themicroorganisms within the bioreactor, preferably about 10% to about 50%,are removed, cycled to a subsequent processing phase, according to anembodiment of the invention, inside or outside the bioreactor, and/oroptionally replaced with fresh growth media or growth media containing alow concentration of microorganisms, in order to allow moremethanotrophic microorganisms to grow or metabolize in the bioreactorand continue to treat the methane within the landfill gas at high rates.

The microorganisms that are removed and/or cycled to a subsequentprocessing phase inside or outside the bioreactor (depending on theembodiment) are processed further according to the specification of theend-product of microorganism growth. For example, if the microorganismbiomass is to be used to generate a polymer such as PHB, themicroorganisms may be exposed to a bioreactor receiving a continuoussupply of landfill gas and air or oxygen, wherein the growth medium isdeprived of a specific essential nutrient, such as nitrogen, in order tocause the microorganisms to synthesize intracellular PHB. After a periodof about 0.1 to about 30 hours, some portion of the bioreactor may thenbe removed in order to harvest the products of bioreactor growth, inthis case PHB. PHB may be harvested through a variety of well knownharvesting, cell extraction, dewatering, and/or polymer purificationtechniques. Dewatering methods may include, but are not limited to, theuse of centrifuges, spray driers, belt filter presses, freeze drying,fluid bed drying, ribbon drying, flocculation, pressing, and/orfiltration. Cell lysis and cell parts separation methods may include,but are not limited to, the use of hot chloroform, sodium hydroxide,cell freezing, sonication, and homogenization. For homogenization, thepressure drop is preferably between about 5000 and about 10,000 bar toeffect sufficient cellular lysis. For the use of sodium hydroxide, theconcentration of sodium hydroxide is preferably raised to approximately2 M. If the microorganism biomass is to be used directly as a proteinsource, the suspended biomass may be dewatered in a belt filter press,bag filter, spray drier, and/or centrifuge, all of which may be used toreduce the water content of the biomass, preferably below about 10% toabout 20% total biomass weight. Isolated, dried, and/or harvestedmicroorganism product, such as biomass, polymer, or enzyme, may be usedor sold for use.

While the above description of preferred systems and methods of carryingout processes in accordance with embodiments of invention contains manyspecificities, these should not be construed as limitations on the scopeof the invention. As stated, there are a number of ways to carry out aprocess in accordance with invention. Accordingly, the scope of theinvention should be determined not by the preferred systems and methodsdescribed, but by the appended claims and their legal equivalents.

1. (canceled)
 2. A method for producing a polyhydroxyalkanoate (PHA) ina culture of methanotrophic microorganisms, the method comprising: a)providing a gas comprising methane and one or more non-methanesubstances; b) providing a culture of methanotrophic microorganisms; c)providing a microorganism culture medium comprising at least a firstessential nutrient and a second essential nutrient; d) exposing saidculture to said gas; e) controlling the concentration of said firstessential nutrient in said culture medium to a concentration sufficientto induce said methanotrophic microorganisms to produce particulatemethane monooxygenase (pMMO) and soluble methane monooxygenase (sMMO);and f) controlling the concentration of said second essential nutrient,wherein said control of said second essential nutrient causes saidmethanotrophic microorganisms to produce said PHA.
 3. The method ofclaim 2, wherein said PHA is selected from the group consisting ofpolyhydroxybutyrate, polyhydroxybutyrate-covalerate (PHBV),poly-4-hydroxybutyrate (P4HB), polyhydroxyhexanoate (PHHx), andpolyhydroxyoctanoate (PHO).
 4. The method of claim 2, wherein said stepe) is followed by said step f), and wherein step e) followed by step f)comprises a production cycle.
 5. The method of claim 4, furthercomprising repeating step e) followed by step f) one or more times,thereby resulting in at least a first production cycle and a secondproduction cycle.
 6. The method of claim 5, wherein the molecular weightof said PHA produced in said first production cycle differs from themolecular weight of said PHA produced in said second production cycle byless than 50%.
 7. The method of claim 6, wherein said molecular weightranges from about 100 to about 5,000,000 Daltons.
 8. The method of claim6, wherein said molecular weight distribution ranges from about 100,000to about 2,500,000 Daltons.
 9. The method of claim 5, wherein thepolydispersity of said PHA produced in said first production cyclediffers from the polydispersity of said PHA produced in said secondproduction cycle by less than 75%.
 10. The method of claim 5, whereinthe polydispersity of said PHA produced in said first production cyclediffers from the polydispersity of said PHA produced in said secondproduction cycle by less than 50%.
 11. The method of claim 9, whereinsaid polydispersity ranges from about 0.1 to about 5.0.
 12. The methodof claim 2, wherein said one or more non-methane substances are selectedfrom the group consisting of methanol, acetone, acetate, formate,formaldehyde, hydroxyalkanoates, hydroxybutyrate, octanoic acid,octanol, carbon dioxide, nitrogen, oxygen, di-oxygen, di-nitrogen,water, water vapor, argon, ethane, propane, butyrate, butyric acid,hexanoic acid, hexanol, heptanoic acid, heptanol, pentane, pentanoicacid, and volatile organic compounds.
 13. The method of claim 2, whereinsaid culture comprises two or more species of methanotrophicmicroorganisms.
 14. The method of claim 2, wherein said first and saidsecond essential nutrients comprise one or more of carbon, hydrogen,nitrogen, oxygen, phosphorus, potassium, calcium, sodium, chlorine,methane, carbon dioxide, magnesium, iron, copper, sulfate, manganese,boron, zinc, aluminum, nickel, chromium, cobalt, or molybdenum.
 15. Themethod of claim 5, wherein the concentration of said pMMO and said sMMOproduced in said microorganisms in said first production cycle differsby less than 75% from the total concentration of said pMMO and said sMMOproduced in said microorganisms in said second production cycle.
 16. Themethod of claim 2, wherein said controlling of said first essentialnutrient or of said second essential nutrient comprises increasing theconcentration of first essential nutrient or of said second essentialnutrient.
 17. The method of claim 2, wherein said controlling of saidfirst essential nutrient or of said second essential nutrient comprisesdecreasing the concentration of first essential nutrient or of saidsecond essential nutrient.
 18. The method of claim 2, wherein the gascomprising methane is produced from a landfill, wherein thepolyhydroxyalkanoate produced is PHB, wherein the culture ofmicroorganisms are exposed to said gas comprising methane in abioreactor, and wherein the culture of methanotrophic microorganismscomprises a non-specified consortium of methanotrophic microorganisms.